CN102965369B - Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof - Google Patents
Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof Download PDFInfo
- Publication number
- CN102965369B CN102965369B CN201210484859.5A CN201210484859A CN102965369B CN 102965369 B CN102965369 B CN 102965369B CN 201210484859 A CN201210484859 A CN 201210484859A CN 102965369 B CN102965369 B CN 102965369B
- Authority
- CN
- China
- Prior art keywords
- soybean
- root rot
- sattwd15
- phytophthora root
- rpswd15
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and an application thereof. A disease-resistant variety Wan pea No.15 is used as a male parent, a susceptible variety Williams (Rps) is used as a female parent, the male parent and the female parent are hybridized to generate a F1 generation, an F2:3 population obtained by selfing of the F1 generation is used as a mapping population, a published soybean SSR (simple sequence repeat) mark and a new SSR mark developed according to the soybean genomic sequence are used for genetic mapping and fine orientation of the disease-resistant gene contained in Wan pea No. 15. The disease-resistant gene Rps WD15-2 is positioned on a No.17 chromosome and is between Sattwd15-24/25 (0.5cM) and Sattwd15-47 (0.8cM), besides, a molecule mark Sattwd15-32 coseparated from the Rps WD15-2 gene is also obtained, and can be used for assistant selection for phytophthora root rot of soybean.
Description
Technical field
The invention belongs to agricultural biotechnology engineering and disease-resistant crops genetic breeding field, specifically, relate to a kind of be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from molecule marker and application.
Background technology
The soybean phytophthora root rot caused by soybean phytophthora (Phytophthora sojae Ka μ fmann & Gerdemann) is one of Major Diseases of harm Soybean production.In recent years, in major soybean production areas Heilongjiang Province, harm becomes increasingly conspicuous this disease, and harm soybean area reaches 15 × 10
4hectare.This disease can work the mischief at any growing stage of soybean, and general field underproduction 10-30%, serious plot can reach 60-90%, even causes total crop failure, causes serious threat to Soybean production.At present, soybean phytophthora root rot has become the Common Diseases threatening soybean in China, so very urgent to the control of this disease.
Facts have proved, to cultivate and plantation disease-resistant variety is the safest, economy and effective measures.For this reason, many scholars have done large quantifier elimination in screening Resistance resource and localization of disease resistance genes.Research shows, the resistance of soybean to phytophthora root rot is divided into qualitative character resistance (microspecies resistance) and quantitative character resistance.Qualitative character resistance is controlled by dominant gene, has complete resistance.So far, on soybean gene group 9 sites, 15 anti-soybean phytophthora root rot genes (Rps) are identified abroad, i.e. Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps 7, Rps8 and theRps gene in cv.Waseshiroge, lays respectively at soybean gene group the 3rd, 16,13,18,18,18, (MLG N, J, F, G on 3,13 and No. 3 karyomit(e)s, G, G, N, F and N) (Sugimoto etc., 2012).The history that soybean phytophthora root rot occurs in China is shorter, and breeding for disease resistance work is also at the early-stage.At present, China only identifies 4 disease-resistant gene RpsYB30, RpsYD25, RpsZS18, RpsSN10 lay respectively at soybean gene group the 19th, and 3, (MLG L, N, D1b and F) (Fan Aiying etc. on 2 and No. 13 karyomit(e)s, 2009, Sun Shi etc., 2011, Yao Haiyan etc., 2010, Yu Anliang etc., 2010, Zhu Zhendong etc., 2007).
China's soybean phytophthora has group structure complexity, and wide accommodation and virulence change the features such as fast, easily produces new virulence type microspecies and overcomes varietal resistance.It is generally acknowledged that the work-ing life of soybean phytophthora root rot gene is 10-15, but the speed that new disease-resistant gene is overcome is accelerated, less than 3 years after as identified in Rps8, just found the soybean phytophthora bacterial strain of the new virulence type that can overcome its resistance.Large quantity research shows, in 15 disease-resistant genes identified (except the Rps gene in cv.Waseshiroge), except Rps1c and Rps1k, not to be highly resistant to the soybean phytophthora population of China lesion abroad.Therefore, be necessary to excavate and utilize new Resistance resource energetically and constantly carry out the cumulative of disease-resistant gene, cultivating the soybean varieties that resistance is lasting.
For excavating new soybean Resistance resource, Most scholars carries out localization of disease resistance genes and molecular marker assisted selection breeding by SSR molecular marker (http://soybase.org).Such mark has the advantages such as high information quantity, reliability are strong, easy and quick, is widely used.In addition utilize the soybean genomic sequence (http://www.Phytozome.net) announced to develop corresponding molecule marker, Fine Mapping can be carried out to gene, obtain the closely linked molecule marker of gene.
The soybean varieties Wan Dou15Shi Anhui Province Pan Cunhu farm institute of agricultural sciences, with covering celebrating 13 systematic breeding after radiation, was authorized in 1996 by Anhui Province's quality.This kind has on resistance (Li Jun mountain 2007) Viruses Infecting Soybean Plant disease and oidium.Early-stage Study result shows, this kind all has stronger resistance (Zhu Zhendong etc., 2001) to two of different virulence type soybean phytophthora bacterial strain PsMC1 and PsMC2.Chen Xiaoling etc. (2008) and summer yataghan etc. (2011) report that beans 15 pairs of soybean phytophthora bacterial strains in Anhui have resistance of wide spectrum again, infer that it may containing new disease-resistant gene.
Summary of the invention
The object of this invention is to provide a kind of be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from molecule marker and application.
In order to realize the object of the invention, of the present invention a kind of be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from molecule marker, wherein, described candidate gene RpsWD15-2 is positioned on soybean No. 17 karyomit(e), and between mark Sattwd15-24/25 and Sattwd15-47, be respectively 0.5cM and 0.8cM with these two genetic distances marked.
Described molecule marker is Sattwd15-32, containing repeating motif (AAC) 6, and for amplifier molecule mark Sattwd15-32 Specific PCR primers to for:
Upstream primer F:5 '-ATCCCTTATTCCCTTCAT-3 ' and
Downstream primer R:5 '-CATAGACCTCCTTCCAAA-3 '.
The genetic distance of molecule marker Sattwd15-32 and candidate gene RpsWD15-2 is 0cM.
The annealing temperature that pcr amplification uses is preferably 47 DEG C, and amplified production size is 149bp.
Wherein, the nucleotide sequence of candidate gene RpsWD15-2 is as shown in SEQ ID NO.1.
The present invention be also provided for detecting to be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from the Specific PCR primers pair of molecule marker Sattwd15-32, comprising:
Upstream primer F:5 '-ATCCCTTATTCCCTTCAT-3 ' and
Downstream primer R:5 '-CATAGACCTCCTTCCAAA-3 '.
The present invention also provide to be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from molecule marker identifying the application in resistant to phytophthora root rot soybean varieties, it comprises step: the genomic dna 1) extracting soybean to be measured; 2) with the genomic dna of soybean to be measured for template, utilize primers F described in claim 4 and R, carry out pcr amplification reaction; 3) detect pcr amplification product, if the product of 149bp can be amplified, be then judged to be resistant to phytophthora root rot soybean varieties.
Wherein, the amplification system that PCR reaction uses is counted with 20 μ l: Soybean genomic DNA template concentrations 2ng/ μ L, 10 × PCR damping fluid 2.5pl, four kinds of dNTP concentration are respectively 20 μm of ol/L, upstream and downstream primer concentration is respectively 0.2 μm of ol/L, Taq archaeal dna polymerase 0.5U, uses ddH
2o complements to 20 μ l.
PCR reaction use condition be: 95 DEG C 3 minutes; 94 DEG C 50 seconds, 47 DEG C 50 seconds, 72 DEG C 50 seconds, 35 circulations; 72 DEG C 10 minutes.
The present invention also provides the test kit for detecting resistant to phytophthora root rot soybean containing above-mentioned primer pair.Preferred described test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, PCR reaction buffer, one or more in standard positive template etc.
The present invention also provide above-mentioned be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from the application of molecule marker in plant molecular marker assistant breeding.
The present invention further provides above-mentioned be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from the application of molecule marker in Soybean Resistance phytophthora root rot molecular breeding.
Particularly, of the present invention be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from molecule marker and candidate gene RpsWD15-2 obtain by the following method:
Utilize the F that Williams × Anhui beans 15 cross combination is derivative
2:3the hereditary basis of Anhui beans 15 pairs of soybean phytophthora root rot resistances, as mapping population, is studied by colony; Utilize the soybean ssr marker announced, the molecule marker that screening is chain with disease-resistant gene; Utilize soybean genomic sequence information, develop new SSR marker, Fine Mapping is carried out to this gene, simultaneously obtain with this gene be divided into from molecule marker.Thus provide assisted Selection for Chinese People's Anti-Japanese Military and Political College's beans resistant to phytophthora root rot breeding, improve breeding efficiency, accelerate resistant to phytophthora root rot breeding work process.
Concrete technical scheme:
(1) resistant analysis of Anhui beans 15
Using disease-resistant variety Anhui beans 15 as male parent, susceptible variety Williams(rps) as maternal, hybridization produces F
1generation, F
1f is produced for selfing
2generation, F
2for 102 F that selfing produces
2: 3the qualification of family resistant analysis, disease-resistant gene and mapping population.Adopt hypocotyl wound inoculation method, resistant analysis is carried out in the reaction of each Parentage determination 20-30 plant to soybean phytophthora bacterial strain PsMC1.At 25 DEG C after inoculation, humidity is under 80-100% condition, and moisturizing 48h, then proceeds to hot-house culture, carries out Disease investigation after 6d.With reference to (2006) method evaluation standards such as Gordon, investigation F
2:3family anti-, be separated, the segregation ratio of susceptible family, the genetics of resistance rule of research Anhui beans 15 pairs of phytophthora root rots.
(2) extraction of gene DNA and anti-sense pond build
CTAB method is adopted to extract 2 parents and 102 F
2:3the genomic dna of family.Each F
2:3family gets 20 single-strain blade samples respectively, and balanced mix extracts DNA.Anti-, the sense pond of 10 disease-resistant, susceptible familys structures for separating of colony's fractional analysis (Bulk Segregating Analysis, BSA) is got according to (1991) such as Michelmore.
(3) SSR marker is utilized to locate the disease-resistant gene of Anhui beans 15
Soybean ssr marker sequence derives from http://soybase.org.Random selecting SSR marker, between Anhui beans 15 and Williams, carries out pcr amplification and polymorphism screening between anti-sense pond.PCR reaction system is 20 μ l, and containing 10 × PCR damping fluid 2.5 μ l, four kinds of dNTPs each 20 μm of ol/L, Taq DNA polymerase 0.5U, upstream and downstream primer is respectively 0.2 μm of ol/L, and template DNA concentration is 2ng/ μ L.
PCR reacts amplification program, 95 DEG C of denaturations 3 minutes, 94 DEG C of sex change 50 seconds, and 47-51 DEG C of annealing 50 seconds, 72 DEG C extend 50 seconds, establish 35 circulations from sex change altogether to extension, last 68 DEG C of extensions 10 minutes, 4 DEG C of preservations.Amplified production carries out electrophoresis and analysis in 8-12% polyacrylamide gel.
(4) Fine Mapping of disease-resistant gene
For making this gene more effectively and more accurately be applied in molecular marker assisted selection breeding work, utilizing the soybean genomic sequence information announced, developing new SSR marker, Fine Mapping is carried out to this gene.Utilizing the SSR marker announced in soybase database by the basis of disease-resistant gene Primary Location, first in Phytozome database (http://www.phytozome.net/soybean) download and this gene linkage two mark zones between sequence, then SSR Hunter 1.3(http is used: //en.bio-soft.net/dna/SSRHunter) find SSR site, finally use software Primer Premier 5.0(Premier Biosoft International, Palo Alto, CA) design SSR primer.PCR reaction system, amplification program and pcr amplification product detection method are with reference to the description in (3).
To the SSR primer with polymorphism, carry out F
2:3colony verifies.At F
2: 3in colony, the SSR primer identical with Anhui beans 15 genotype is designated as AA, and the SSR primer identical with Williams genotype is designated as BB, and heterozygous genotypes is designated as AB.
(5) data analysis
To F
2:3population resistance separation and SSR marker are at F
2:3clastotype in family carries out χ
2inspection.With Joinmap 4.0(Van Ooijen 2006) linkage relationship of software analysis SSR marker and disease-resistant gene.
Soybean phytophthora root rot is a kind of serious plant disease affecting Soybean production.The present invention utilizes SSR molecular marker method, on soybean No. 17 karyomit(e), located a new Resistant candidate genes RpsWD15-2 first and obtain to be divided into this disease-resistant gene RpsWD15-2 from and closely linked molecule marker.This disease-resistant gene has resistance of wide spectrum, effectively can control the generation of China's soybean phytophthora root rot, alleviate the production loss that this disease causes.Utilize to be divided into this gene from SSR marker Sattwd15-32, effectively can be applied in molecular marker assisted selection breeding work, overcome cycle required for conventional breeding methods long shortcoming; PCR-based technology, carries out Analysis of Resistance in indoor to soybean resource; Simultaneously for this gene of clone lays the foundation, and carry out structure and function analysis to it, this is significant for the molecular genetics mechanism understanding this gene further, puts into practice, has very important value in breeding work and disease-resistant theoretical investigation at Soybean production.Its advantage is:
(1) the present invention located a new anti-soybean phytophthora root rot candidate gene RpsWD15-2 first on soybean No. 17 karyomit(e).This gene is in the soybean phytophthora bacterial strain PsMC1(virulence type stronger to China's virulence: 1a, 1c, 1k, 2,3b, 3c, 4,5,6,7,8, ZS18) there is acquisition in the soybean varieties Anhui beans 15 of complete resistance.Meanwhile, this gene pairs China soybean phytophthora has resistance of wide spectrum, effectively can control the generation of China's soybean phytophthora root rot, alleviate the production loss that this disease causes.
(2) of the present invention be divided into candidate gene RpsWD15-2 from molecule marker Sattwd15-32, the new mark obtained in the filial generation family of Anhui beans 15 and resistance thereof, the molecular marker assisted selection breeding in may be used for soybean phytophthora root rot cultivar identification and Soybean Resistance generation after being ill.
(3) of the present invention be divided into candidate gene RpsWD15-2 from molecule marker Sattwd15-32, for the Cloning and sequencing of this gene lays the foundation, thus clearly this sequence information and constitutional features.Meanwhile, according to be divided into from molecule marker and soybean genomic sequence information, carry out the haplotype analysis of this gene, be expected to be cloned into new disease-resistant gene.
Accompanying drawing explanation
Fig. 1 is after inoculating soybean phytophthora PsMC1 bacterial strain 6d in the embodiment of the present invention 1, the growing state of Anhui beans 15 and Williams.
Fig. 2 is soybean phytophthora root rot candidate gene RpsWD15-2 and chain SSR marker genetic linkage map thereof in the embodiment of the present invention 1.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The candidate gene RpsWD15-2 that embodiment 1 Soybean Resistance phytophthora root rot is new and be divided into from the acquisition of molecule marker Sattwd15-32
The resistant analysis of 1.1 Anhui beans 15
Using disease-resistant variety Anhui beans 15 as male parent, susceptible variety Williams(rps) as maternal, preparing hybrid combination produces F
1generation, F
1f is produced for selfing
2generation, F
2for 102 F that selfing produces
2: 3family is as resistant analysis, disease-resistant gene qualification and mapping population.Adopt hypocotyl wound inoculation method, each Parentage determination 20-30 plant carries out resistant analysis to the reaction of bacterial strain PsMC1.At 25 DEG C after inoculation, humidity is under 80-100% condition, and moisturizing 48h, then proceeds to hot-house culture, carries out Disease investigation after 6d.With reference to Gordon(2006) etc. method evaluation standard, namely plant mortality ratio is less than the family of 20% is pure and mild disease-resistant family, and the family that mortality ratio is greater than 80% is pure and mild susceptible family, and the family that mortality ratio is 21-79% is Resistant segregation family, investigation F
2:3family anti-, be separated, the segregation ratio of susceptible family, the genetics of resistance rule of research Anhui beans 15 pairs of phytophthora root rots.(Fig. 1)
The F that 102 Williams × Anhui beans 15 cross combination is derivative
2:3the Resistance Identification result of family to bacterial strain PsMC1 shows, 31 familys are pure and mild disease-resistant, and 42 familys are heterozygous, and 29 familys are susceptible, through χ
2inspection is separated the 1:2:1 meeting expectation, shows that the resistance of Anhui beans 15 couples of soybean phytophthora bacterial strain PsMC1 is by a dominant Dominant gene, by this candidate gene called after RpsWD15.
The extraction of 1.2 Soybean genomic DNAs and anti-sense pond build
CTAB method is adopted to extract 2 parents and 102 F
2:3the genomic dna of family.Each F
2:3family gets 20 single-strain blade samples respectively, and balanced mix puts into the centrifuge tube of 2.0mL.Be placed in liquid nitrogen to be ground into powder, add the CTAB Extraction buffer of 800 μ L, 65 DEG C of preheatings, be incubated 30-50 minute after mixing in 65 DEG C of water-baths in centrifuge tube, shake is in order to avoid agglomerating frequently; Take out centrifuge tube, be cooled to room temperature, add 800 μ L phenol: chloroform: primary isoamyl alcohol (25:24:1), extracting 10 minutes, period puts upside down at set intervals gently, makes it fully mix, centrifugal 10 minutes of 12000rpm; Get supernatant liquor in another 1.5mL centrifuge tube, add equal-volume chloroform: primary isoamyl alcohol (24:1), fully mixing is after 10 minutes, centrifugal 10 minutes of 12000rpm; Get supernatant liquor in another 1.5mL centrifuge tube, add the Virahol of 0.8 times of volume precooling, slowly mix, at-20 DEG C, place 30-60 minute, make DNA with Precipitation; Choosing DNA precipitation is transferred in 1.5mL centrifuge tube, with 75% ethanol wash 2 times, finally dewaters with dehydrated alcohol, dries up, add the ultrapure water of appropriate sterilizing.After DNA dissolves, the agarose gel electrophoresis with 0.8% detects the integrity of DNA and the concentration of ultraviolet spectrophotometry detection DNA sample, DNA concentration is adjusted to 40ng/ μ L.
Anti-, the sense pond for separating of colony's fractional analysis (Bulk Segregating Analysis, BSA) is built according to methods such as (1991) such as Michelmore.The DNA respectively getting 10 disease-resistant familys of 1 μ L is mixed in a new centrifuge tube, is built into anti-pond, and DNA concentration is adjusted to 40ng/ μ L.Sense pond construction process is with reference to anti-pond construction process.
1.3 utilize SSR marker to locate the disease-resistant gene of Anhui beans 15
Soybean ssr marker sequence derives from http://soybase.org.Random selecting SSR marker carries out pcr amplification and polymorphism screening between Anhui beans 15 and Williams and between anti-sense pond.
PCR reaction system is 20 μ l, and containing 10 × PCR damping fluid 2.5 μ l, four kinds of dNTPs each 60 μm of ol/L, Taq DNA polymerase 0.5U, upstream and downstream primer is respectively 0.2 μm of ol/L, and template DNA concentration is 2ng/ μ L.
PCR reacts amplification program, 95 DEG C of denaturations 3 minutes, 94 DEG C of sex change 50 seconds, and 47-51 DEG C of annealing 50 seconds, 72 DEG C extend 50 seconds, establish 35 circulations from sex change altogether to extension, last 72 DEG C of extensions 10 minutes, 4 DEG C of preservations.
PCR primer detects, and according to the ratio of 1:5, joins in PCR primer by the 6 × sample-loading buffer of 4 μ l, and after mixing, amplified production carries out electrophoresis and analysis in 8-12% polyacrylamide gel.
SSR marker on random selecting soybean, finds between two parents, to have polymorphism from 21 pairs of SSR marker on No. 17 karyomit(e).These 21 marks are used for anti-sense pond and detect polymorphism, wherein only have 9 to mark and produce polymorphism, they may be chain with disease-resistant gene.Use the F that 102 Williams × Anhui beans 15 cross combination is derivative further
2: 3family carries out chain checking to these 9 marks, and result shows that 9 the mark Sat 222, Sat_292, Satt514, Satt461, Satt528, Satt574, Satt543, Satt615 and Satt301 and RpsWD15 derived from No. 17 karyomit(e) are chain.These 9 are marked at all distributions in 1:2:1 in colony, are codominant marker.Show with Joinmap 4.0 software analysis, RpsWD15-2 gene and these 9 mark chain, and between mark Satt543 and Satt615, are respectively 2.2cM and 7.2cM with these two genetic distances marked.
The Fine Mapping of 1.4 disease-resistant genes
For making this gene more effectively and more accurately be applied in molecular marker assisted selection breeding work, utilizing the soybean genomic sequence announced to develop new SSR marker, Fine Mapping is carried out to this gene.In Phytozome database, (http://www.phytozome.net/soybean) compare of analysis is known, and between Satt543 and Satt615, physical distance is 4.29Mb and downloads this sector sequence; Search out 671 SSR sites altogether with SSR Hunter 1.3 (http://en.bio-soft.net/dna/SSRHunter), being wherein greater than 10 SSR sites of repeating motif has 414; On these 414 SSR sites, primer is designed, middle acquisition 414 SSR marker with software Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA).PCR reaction system, amplification program and detection method are with reference to the description in (3).
Adopt BSA method, obtain the SSR marker of 17 polymorphisms altogether, may be chain with disease-resistant gene.Use the F that 102 Williams × Anhui beans 15 cross combination is derivative further
2: 3family carries out chain checking (table 1) to these 17 marks.Result shows, these 17 are marked at all distributions in 1:2:1 in colony, are codominant marker.
Table 1 is according to soybean genomic sequence design and the Molecular Marker Information chain with candidate gene RpsWD15-2
Show with Joinmap 4.0 software analysis, RpsWD15 gene and these 26 mark chain, and between mark Sattwd15-24/25 and Sattwd15-47, are respectively 0.5cM and 0.8cM with these two genetic distances marked.Meanwhile, also obtain with RpsWD15-2 candidate gene be divided into from molecule marker Sattwd15-32, mark Sattwd15-32 and candidate gene RpsWD15-2 genetic distance be 0cM.(Fig. 2).
Known in the comparison of Phytozome database inner analysis, the genetic model (table 2) having 8 to predict between mark Sattwd15-24/25 and Sattwd15-47, two genetic model Glyma17g28950.1 with Glyma17g28970.1 are wherein had to encode the albumen of the serine/threonine structure relevant to plant disease-resistant, and in the present invention, with disease-resistant gene be divided into from molecule marker Sattwd15-32 be the primer designed according to genetic model Glyma17g28970.1.Therefore Glyma17g28970.1 may be the candidate gene of Anhui beans 15, i.e. called after RpsWD15-2.
On table 2 soybean No. 17 karyomit(e), the genetic model between mark Sattwd15-24/25 and Sattwd15-47
According to 5 '-UTR and the 3 '-UTR design primer (upstream primer: 5 '-CTAACGACCTTTACCCAC-3 ' of genetic model Glyma17g28970.1; Downstream primer: 5 '-ATTTCCACCGAACATACTA-3 '), with Anhui beans 15 genome for template carries out pcr amplification.The object fragment the obtained TIANgel Midi PurificationKit of TIANGEN company, reference reagent box specification sheets reclaims object fragment.
Reclaim the pMD18-T test kit (TaKaRa Code:D101A) that the object fragment obtained adopts TaKaRa company, object fragment is connected with pMD18-T carrier and transforms by reference reagent box specification sheets, and concrete operation step is as follows:
(1) in Eppendorf tube, prepare 5 μ lDNA solution, comprise the pMD18-T carrier of 1 μ l, the Insert Fragment of 3 μ l, the ddH of 1 μ l
2o.
(2) Solution 1 of 5 μ l is added.
10 μ l, after 10 hours, are connected product and join in the JM109 competence intestinal bacteria of 100 μ l, place 30 minutes in ice by (3) 16 DEG C of reactions.
(4) 42 DEG C are heated 45 seconds, then place 1 minute in ice.
(5) add the LB substratum of 600 μ l, 37 DEG C shake cultivation 45 minutes.
(6) cultivate on the LB solid medium containing X-gel, IPTG and Amp, form single bacterium colony.
(7) the single white colony of picking, and after concussion is cultivated in containing the liquid nutrient medium of Amp, confirm the size of Insert Fragment in carrier by PCR method.Sequencing result shows, the nucleotide sequence of Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 is as shown in SEQ ID NO.1, and CDS sequence is as shown in SEQ ID NO.2, and the aminoacid sequence of its coding is as shown in SEQ ID NO.3.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. with Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 be divided into from molecule marker, it is characterized in that, described candidate gene RpsWD15-2 is positioned on soybean No. 17 karyomit(e), and between mark Sattwd15-24/25 and Sattwd15-47, be respectively 0.5cM and 0.8cM with these two genetic distances marked;
Described molecule marker is Sattwd15-32, containing repeating motif (AAC) 6, and for amplifier molecule mark Sattwd15-32 Specific PCR primers to for:
Upstream primer F:5 '-ATCCCTTATTCCCTTCAT-3 ' and
Downstream primer R:5 '-CATAGACCTCCTTCCAAA-3 ';
Wherein, for amplification label Sattwd15-24 Specific PCR primers to for:
Upstream primer: 5 '-CTTTGTCCCCTCCTTTAG-3 '
Downstream primer: 5 '-TTCAACAAGAAAAGGTAA-3 '
For amplification label Sattwd15-25 Specific PCR primers to for:
Upstream primer: 5 '-TCATCCAACAACACGCCATT-3 '
Downstream primer: 5 '-CTCCATAGTTTGCTTTTA-3 '
For amplification label Sattwd15-47 Specific PCR primers to for:
Upstream primer: 5 '-GAACCTAAACCCACCCAA-3 '
Downstream primer: 5 '-TGCTAAAAGGGTGGGAAT-3 '.
2. molecule marker according to claim 1, is characterized in that, the annealing temperature that pcr amplification uses is 47 DEG C, and amplified production size is 149bp.
3. molecule marker according to claim 1, is characterized in that, the nucleotide sequence of described candidate gene RpsWD15-2 is as shown in SEQ ID NO.1.
4. for detect to be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 from the PCR primer pair of molecule marker Sattwd15-32, it is characterized in that, comprising:
Upstream primer F:5 '-ATCCCTTATTCCCTTCAT-3 ' and
Downstream primer R:5 '-CATAGACCTCCTTCCAAA-3 '.
5. be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 described in claim 1 from molecule marker identifying the application in resistant to phytophthora root rot soybean varieties, it comprises step:
1) genomic dna of soybean to be measured is extracted;
2) with the genomic dna of soybean to be measured for template, utilize primers F described in claim 4 and R, carry out pcr amplification reaction;
3) detect pcr amplification product, if the product of 149 bp can be amplified, be then judged to be resistant to phytophthora root rot soybean varieties.
6. application according to claim 5, it is characterized in that, step 2) in PCR reaction use amplification system count with 20 μ l: Soybean genomic DNA template concentrations 2ng/ μ L, 10 × PCR damping fluid 2.5 μ l, four kinds of dNTP concentration are respectively 20 μm of ol/L, upstream and downstream primer concentration is respectively 0.2 μm of ol/L, Taq archaeal dna polymerase 0.5U, uses ddH
2o complements to 20 μ l.
7. application according to claim 5, is characterized in that, step 2) in PCR reaction use condition be: 95 DEG C 3 minutes; 94 DEG C 50 seconds, 47 DEG C 50 seconds, 72 DEG C 50 seconds, 35 circulations; 72 DEG C 10 minutes.
8. the test kit for detecting resistant to phytophthora root rot soybean containing primer pair described in claim 4.
9. test kit according to claim 8, is characterized in that, described test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, PCR reaction buffer, one or more in standard positive template.
10. be divided into Soybean Resistance phytophthora root rot candidate gene RpsWD15-2 described in claim 1 from the application of molecule marker in Soybean Resistance phytophthora root rot molecular breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210484859.5A CN102965369B (en) | 2012-11-23 | 2012-11-23 | Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210484859.5A CN102965369B (en) | 2012-11-23 | 2012-11-23 | Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102965369A CN102965369A (en) | 2013-03-13 |
| CN102965369B true CN102965369B (en) | 2015-01-14 |
Family
ID=47795805
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210484859.5A Expired - Fee Related CN102965369B (en) | 2012-11-23 | 2012-11-23 | Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102965369B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110273022B (en) * | 2019-08-01 | 2023-07-18 | 安阳市农业科学院 | Molecular marker, primer, detection method and application for detecting soybean phytophthora root rot resistance gene |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101677516A (en) * | 2007-04-20 | 2010-03-24 | 孟山都技术公司 | Methods and compositions for selecting soybean plants resistant to phytophthora root rot |
-
2012
- 2012-11-23 CN CN201210484859.5A patent/CN102965369B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101677516A (en) * | 2007-04-20 | 2010-03-24 | 孟山都技术公司 | Methods and compositions for selecting soybean plants resistant to phytophthora root rot |
Non-Patent Citations (1)
| Title |
|---|
| 大豆抗疫霉根腐病基因分析及大豆品种皖豆15抗疫霉根腐病基因作图;夏长剑;《中国优秀硕士学位论文全文数据库农业科技辑》;20111019;D046-45 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102965369A (en) | 2013-03-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103305510B (en) | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof | |
| CN106868131B (en) | SNP molecular marker of upland cotton No. 6 chromosome related to fiber strength | |
| CN104073487B (en) | The molecule marker of rice blast resistant gene Pi2 and application thereof | |
| CN102162011B (en) | Molecule marking method of rice blast-resisting gene | |
| Jia et al. | Identification of a new locus, Ptr (t), required for rice blast resistance gene Pi-ta–mediated resistance | |
| CN104726450B (en) | With the capsicum root-rot epidemic disease closely linked molecule marker of specific resistance gene and application thereof | |
| CN102220430B (en) | Auxiliary screening method for stripe rust-resistance wheat and its special primers | |
| CN102154471B (en) | Molecular marking method for major quantitative trait loci(QTL) for rice grain length | |
| CN101294161B (en) | Molecular mark interlinked with whitebacked planthopper resistance genes of rice, and developing method thereof | |
| KR101922420B1 (en) | Composition comprising DNA marker derived from Nampyeongbyeo for selecting rice variety resistant to bakanae disease and method of selecting rice variety resistant to bakanae disease using the DNA marker | |
| CN1079114C (en) | Nucleic acid markers for rice blast resistance genes and rice blast resistance genes isolated by the use of these markers | |
| CN110512025A (en) | A kind of molecular labeling and its application with powdery mildew resistance gene in wheat PmJM23 close linkage | |
| CN106148510A (en) | Resistance gene of rice blast Pi5 specific Function molecular marker and application thereof | |
| Feng et al. | Molecular mapping of Yr85 and comparison with other genes for resistance to stripe rust on wheat chromosome 1B | |
| CN103952403B (en) | The closely linked molecular marker of rice bacterial blight resistance new gene Xa39 | |
| CN111926104B (en) | SSR molecular marker for identifying authenticity of sugarcane and festuca arundinacea filial generation and method thereof | |
| CN107058519B (en) | Molecular marker CAPs1516 closely linked with soybean anti-epidemic gene and application thereof | |
| CN113528703A (en) | Development and application of KASP molecular marker of rice blast resistance gene Pid3-A4 | |
| CN102994498B (en) | Molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 and application of molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 | |
| CN102965369B (en) | Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof | |
| CN112746123B (en) | Molecular marker closely linked with wheat root rot vermicular spore black embryo disease resistance QTL and application thereof | |
| CN104031995B (en) | A kind of method of molecular marker auxiliary breeding for disease resistance | |
| CN103789431B (en) | Assisting sifting and cultivate the primer special of stripe rust resisting wheat new lines, reagent and test kit and selection thereof | |
| CN103740856B (en) | Molecular method for predicting verticillium wilt resisting capability of different cotton materials | |
| CN102676515B (en) | Lophopyrum elongatum genome-specific molecular markers and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150114 Termination date: 20151123 |